PROJECT SUMMARY/ABSTRACT Nicotine abuse and addiction represent a substantial burden to public health. Nicotine, an active alkaloid in tobacco, is responsible for addiction to tobacco-containing products such as cigars, cigarettes, and vaporized liquid e-cigarettes. Given the immense negative health impact of nicotine addiction as well as the recent surge in popularity of nicotine-containing e-cigarettes, there is a great need for innovative research on the neurobiological underpinnings of nicotine addiction and relapse. Nicotine produces cellular adaptations in brain regions associated with drug reward, such as the nucleus accumbens (NA). However, the neurobehavioral mechanisms underlying the development of addiction and relapse vulnerability are relatively unknown. Nucleus accumbens core (NAcore) glutamatergic mechanisms are involved in nicotine relapse, including rapid, transient potentiation of synaptic strength (measured as increased dendritic spine diameter and AMPA currents) and accompanying glutamate receptor changes. Interestingly, nicotinic acetylcholine receptors (nAChRs), specifically ?7 and ?2-containing, mediate this rapid, transient nicotine relapse-associated plasticity within the NAcore. The principal hypothesis of the proposed studies is that cue-induced glutamate release at prefrontal cortex (PFC)-NA projections (specifically, the prelimbic (PL) subregion of the PFC, and the NAcore) activates release of acetylcholine (ACh) from NA cholinergic interneurons (ChIs) that feeds forward to promote prolonged glutamate release contributing to relapse of nicotine-seeking behavior. In this way, ChIs may modulate aberrant glutamatergic signaling by exacerbating the signal and thus unlocking the transition from drug craving to seeking. Due to the cue dependency of smoking behavior, exposure to nicotine-associated cues is a risk factor for relapse. In the proposed studies, we will explore ChI-specific mechanisms modulating both cued nicotine reinstatement and associated changes in synaptic plasticity, and perform a circuit-level analysis of neuronal interactions underlying relapse vulnerability. In Specific Aim 1, we will examine the impact of ChI signaling on prefrontal cortex glutamatergic projections into the accumbens core, and the impact of this subcircuit on cue-induced nicotine seeking and ACh release. In Specific Aim 2, ChIs will be chemogenetically activated or inhibited within the accumbens core and rapid, transient synaptic plasticity in medium spiny neurons (MSNs) associated with nicotine seeking behavior will be measured. We predict that manipulation of ChIs will result in altered relapse- associated synaptic plasticity in MSNs, which in turn will lead to alterations in nicotine seeking motivation. In both aims, novel techniques will be utilized from multiple levels of analysis including optogenetics, chemogenetics, microdialysis, behavior, and electrophysiology. In conclusion, findings from these investigations will extend to future goals of defining the neural circuitry and neurobehavioral mechanisms underlying nicotine addiction- related behaviors.